biochem (chapt.5)

Chapter 5.Metabolism of Lipids

Lipids

Insoluble or immiscible

Triacylgerols

store and supply energy for metabolism. Lipoids: phospholids, glycolipids, cholesterol and

cholesterol ester

membrane components

Metabolism of lipid Fatty acids

esterified to some backbone molecules

glycerol sphingosine

cholesterol

Metabolism of Lipids Fats

store in adipose tissue

Essential fatty acids: formation of membrane, regulation of chollesterol metabolism, precursors of eicosanoids (protaglandins, thromboxanes and leukotrienes.

Necessary unsaturated fatty acids

Fat Facts Dietary lipids are 90% triacylglycerols; also include

cholesterol esters, phospholipids, essential unsaturated fatty acids; fat soluble vitamins (A,D,E,K)

Fat is energy rich and provides 9 kcal/gm

Normally essentially all (98%) of the fat consumed is absorbed, and most is transported to adipose for storage.

SIX STEPS OF LIPID DIGESTION AND ABSORPTION

Minor digestion of triacylglycerols in mouth and stomach by lingual (acid-stable) lipase

Major digestion of all lipids in the lumen of the duodenum/jejunum by pancreatic lipolytic enzymes

Bile acid facilitated formation of mixed micelles that present the lipolytic products to the mucosal surface, followed later by enterohepatic bile acid recycling

Assembly and export from intestinal cells to the lymphatics of chylomicrons coated with Apo B48 and containing triacylglycerols, cholesterol esters and phospholipids

Passive absorption of the lipolytic products from the mixed micelle into the intestinal epithelial cell

Reesterification of 2-monoacylglycerol, lysolecithin, and cholesterol with free fatty acids inside the intestinal enterocyte

Lipase Site of Action

Regulation Preferred Substrate

Ccleaved

Product(s)

lingual/acid-stable lipase

mouth, stomach

---- TAGs with med. chain FAs

3 FFA+DAG

pancreatic lipase

small intestine

colipase (+) TAGs with long-chain FAs

1 and 3 FFA+2MG

milk lipase small intestine

bile acids (+) TAGs with med. chain FAs

1 and 2 and 3

FFA+glycerol

phospholipase A2 (PLA2)

small intestine

bile acids (+) Ca2+ (+)

PLs with unsat. FA on position 2

2 Unsat FFA lysolecithin

lipoprotein lipase

capillary walls

apo CII (+)insulin (+)

TAGs in chylo-micron or VLDL

1 and 2 and 3

FFA+glycerol

Hormone-sens. Lipase

adipose cell insulin (-)glucagon (+)Epineph. (+)

TAG stored in adipose cells

3 FFA+DAG

Summary of the physiologically important lipases

Absorption of Lipids

Metabolism of Triacylglyerols

Mobilization of fats from triacylglycerols

Hormone sensitive lipase

Rate-determining step

Specific for removing first fatty acid

Phosphorylated form is active

LIPOLYSIS

RECEPTORS

proteinkinase A

cellmembrane

EpinephrineGlucagon

HORMONES

cyclicAMP

= activation- = inhibition

TriacylglycerolFatty acid +

Diacylglycerol

OPHSL-a

HSL = hormone-sensitive lipase

proteinphosphatase

+ Insulin

- caffeinetheophylline

phosphodiesterase

Figure 1. Hormonal activation of triacylglycerol (hormone-sensitive) lipase. Phosphorylation brings about activation to HSL-a.

Adenylylcyclase

(inactive form)

HSL-bOH

inactive

active

lipolysis Glycerols and fatty acids

diffuse out of adipose cells and enter into circulation

Free fatty acids (FFA)

form fatty acid-albumin complexes Glycerols

to form dihydroxyacetone phosphate (DHAP)

Figure. Page 176

Beta-Oxidation of Fatty Acids

Beta Oxidation Part IThe break down of a fatty acid to acetyl-CoA

units…the ‘glycolysis’ of fatty acids

Occurs in the mitochondria

Exemplifies Aerobic Metabolism

at its most powerful phase

STRICTLY AEROBIC

Acetyl-CoA is fed directly into the Krebs cycle

Overproduction causes KETOSIS

CH3CH2CH2COOH

[CH3CH2CH2CO-AMP]

CH3CH2CH2CO~SCoA

HS-CoA

Fatty acyl CoA

AMPAcyl-CoA synthetase

Prepares a Fatty Acid for transport and metabolism

CH2CH2CH2COO CH2CH2COO

OCH2CO COO

(even chain) (odd chain)

Phenylpyruvate Benzoate

Knoop’s Experiment

Phenylacetate Benzoate

Beta-Oxidation of Fatty Acids

C6H12O6 + 6O2 6CO2 + 6H2O Ho = -2,813 kJ/mol = - 672 Cal/mol = 3.74 Cal/gram

C18H36O2 + 26O2 18CO2 + 18 H2O Ho = -11,441 kJ/mol = - 2,737 Cal/mol = 9.64 Cal/gram

THE ENERGY STORY

Glucose

Stearic Acid

On a per mole basis a typical fatty acid is 4 times more energy rich that a typical hexose

Sample calculation of energy produced for the cell via -oxidation of palmitate (a C16 fatty acid):

Palmitoyl-CoA

Palmitoyl-CoA + 7CoA + 7FAD + 7NAD+ + 7H2O

8 Acetyl-CoA 80 ATP

7 FADH2 10.5 ATP

7 NADH + 7H+ 17.5 ATP

108 ATP-2 ATPTotal 106 ATP

Beta Oxidation Part II

Unsaturated fatty acid

Polyunsaturated fatty acid

Odd number chain fatty acid

Obstacle of cis double bonds

Obstacle of position of double bond

Obstacle of 3 carbons at the end

3 Obstacles

CH3CH2CH2CH2CH2C CH2CH2CH2CH2CH2CH2CH2CO~SCoAC=C

HH4 3 2 1

Whoops!A cis D.B. will interfere

Oleic Acid

CH3CH2CH2CH2 CH2 CH2CH2CH2CH2CH2CH2CH2CO~SCoAC=C

HH12345

Linoleic

C18:cis9

Unsaturated and Polyunsaturated Require Additional Enzymes

CH3CH2CH2

C=CCH2CH2CH2-CO~SCoA

Cleavage here

New COO group

New carbon

CH3CH2CH2

CH2C C-CO~SCoA

Enoyl CoAIsomerase

Trans double bond

CH2-CH2

CH2-CH2-CH2-CH2-CH2-CH2CH2C~SCoA

H 9HHH

CH2C~SCoA

CH2C~SCoAO

CH2C~SCoA

CH3C~SCoA12

CH3C~SCoA34

CH3C~SCoA56

Linoleic Acid C18 cis 9,12

Poly Unsaturated (Continued)

-CH2 CH2 CH2CO~SCoAC=CH H

C=CH H

-CH2 CH2 C-CO~SCoAC-CH H

C=CH H

-CH2 CH2

CH2CO~SCoAC=CH H

Enoyl-CoAisomerase

Round 5starter

Round 4starter

Beta carbon to be

Round 5starter

CO~SCoAC=CH H

Dead end

Acyl-CoAdehydrogenase

-CH2 CH2

CH2CO~SCoAC=CH H

New Strategy

C-CO~SCoAC

C=CH H

beta 6

NADPH + H+

2,4 dienoyl-CoAreductase

3,2 enoyl-CoAisomerase

CH2CO~SCoA H

Continue Beta Oxidation

Reduce near (bond), Shift far (bond)

C-CO~SCoAC

C=CH H

beta 6

C-CO~SCoA C

CH2-CH2

beta 6

Ketone bodies formation and utilization

What is Ketosis?An excessive production of ketones in the blood

3 derivatives of acetyl-CoA

Acetoacetate

-hydroxybutyrate

Acetone

CH3CCH2COO-

CH3-C-CH3

CH3CCH2COO-

What is the Significance of ketosis

Acidosis

Excessive acid in the blood

Overflow

Excessive oxidation of fatty acids

Faulty Carbohydrate Metabolism

Metabolic Problem

Metabolic fate of Acetyl CoA

Acetyl-CoA

Pyruvate

Citrate

Ketone BodiesFatty Acidsminor

CH3C~SCoA

OCH3C~SCoA

-Ketothiolase

HS-CoA

CH2C~SCoA

CH3CCH2C~SCoA

rearrangement

Acetoacetyl-CoA

CH3CCH2C~SCoA

O OCH3C~SCoA

CH3CCH2C~SCoA

CH2C-O-

HS-CoA

OOC-CH2-C-CH2-C~SCoA

HMG-CoASynthase

-hydroxy--methylglutaryl-CoA

(HMG-CoA)

OOC-CH2-C-CH2-C~SCoA

CH3-C~SCoA

OOC-CH2-C-CH3

CH3-C-CH3

OOC-CH2-CH-CH3

NADH + H+

Acetoacetate

Acetone-hydroxybutyrate

HMG-CoA

HMG-CoALyase

Utilization of ketone bodies

1. Acetoacetate/succinyl-CoA CoA transferase

2. Acetoacetyl-CoA thiokinase

3. Acetoacetyl-CoA thiolase

Pysiological Significance of ketogenesis

Ketone bodies produced by the liver are excellent fuels for a variety of extrahepatic tissues, especially during times of prolonged starvation.

Reconversion of ketone bodies to acetyl-CoA inside the mitochondria provides metabolic energy.

Regulation of Ketogenesis

Feeding status In the hungry state, higher glucagon and other

lipolytic hormones trigger the lipolytic process in adipose tissue with the result that free fatty acids pass into the plasma for uptake by liver and other tissues. This promotes fatty acid oxidation and ketogenesis in the liver.

Regulation of Ketogenesis Metabolism of glycogen in the hepatic cells

once fats enter the liver, they have two distinct fates: activated to acyl-Co-A and oxidized, or esterified to glycerol in the production of triacylglycerols in cytoplasm. If the liver has sufficient supplies of glycerol-3 phosphate by glucose metabolism, most of the fats will be turned to the production of triacylglycerols. In contrast, glucose deficiency will cause a lower triacylglycerols and ATP generation, with the majority of the FAs entering beta-oxidation leading to a increased production of ketone bodies.

Regulation of Ketogenesis The fall in malonyl-CoA concentration can terminate

the inhibition on carnitine acyltransferase I, such that long-chain fatty acids can be transported through the inner mitochondrial membrane to the enzymes of fatty acid oxidation and ketogenesis. This may happen during a hungry state. In contrast, administration of food after a fast, or of insulin to the diabetic subject, reduces plasma free fatty acid concentrations and increases liver concentration of malonyl-CoA, this will inhibit carnitine acyltransferase I and thus reverses the ketogenic process.

Fatty Acid Biosynthesis Not exactly the reverse of degradation

by a different set of enzymes , in a different part of the cell

Primarily in the cytoplasm of the following tissues: liver, kidney, adipose, central nervous system and lactating mammary gland

Liver is the major organ for fatty acid synthesis

LIPID BIOSYNTHESIS Fatty acid biosynthesis-basic fundamentals Fatty acid biosynthesis-elongation and

desaturation Triacylglycerols Phospholipids Cholesterol Cholesterol metabolism

Fatty Acid Biosynthesis

Cytosol Requires NADPH Acyl carrier protein D-isomer CO2 activation

Keto saturated

Mitochondria NADH, FADH2

CoA L-isomer No CO2

Saturated keto

Beta OxidationSynthesis

Fatty acid biosynthesis is a stepwise assemblyof acetyl-CoA units (mostly as malonyl-CoA) ending with palmitate (C16 saturated)

Activation

Elongation

Termination

3 Phases

CH3C~SCoA

ACTIVATION

-OOC-CH2C~SCoA

SCH2CH2CH2CH2CO

NHCH2CH2CH2CH2 ENZYME

SCH2CH2CH2CH2CO

Carboxybiocytin

Biotin

active carbon

Acetyl-CoA carboxylase

Biocytin

Cofactor

ADP + Pi

Acetyl-CoA CarboxylaseThe rate-controlling enzyme of FA synthesis

In Bacteria -3 proteins (1) Carrier protein with Biotin (2) Biotin carboxylase (3) Transcarboxylase

In Eukaryotes - 1 protein (1) Single protein, 2 identical polypeptide chains

(2) Each chain Mwt = 230,000 (230 kDa)(3) Dimer inactive (4)

Activated by citrate which forms filamentous form of protein that can be seen in the electron microscope

Yeast Fatty Acid Synthase Complex

2,500 kDa Multienzyme Complex

6 molecules of 2 peptide chains called A and B

(66)A: (185,000) Acyl Carrier protein -ketoacyl-ACP synthase (condensing enzyme) -ketoacyl-ACP reductase

B: (175,000) -hydroxy-ACP dehydrase enoyl-ACP reductase palmitoyl thioesterase

Fatty AcidSynthaseComplex

Acyl carrier protein10 kDa

Cysteamine

Phosphopantetheine

HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-P-O-CH2

HO CH3

OO Adenine

O-P-OO

Coenzyme A

Acyl Carrier ProteinAcyl Carrier Protein

HS-CH2-CH2-N-C-CH2-CH2-N-C-C-C-CH2-O-P-O-CH2-Ser-O O O

HO CH3

Overall Reaction

CH3C~SCoA

CH2C~S-

HS-CoACO2

Malonyl-CoA carbons become new COOH end

Nascent chain remains tethered to ACP

Acyl CarrierProtein

CO2, HS-CoA are released at each condensation

Malonyl-CoA + ACP

-OOC-CH2C~S-

ACP + HS-CoA

Initiation

CH2C~S-

CH3CH2CH2C~S-

CH3C- CH2C~S-

CH3C- = C- C~S-

-Carbon Elongation

D isomer

Reduction

Dehydration

Reduction

-Ketoacyl-ACP reductase

-Hydroxyacyl-ACP dehydrase

Enoyl-ACP reductase

-S-ACP

TERMINATION Ketoacyl ACPSynthase

Free to bindMalonyl-CoA

Transfer to KS

Split out CO2

Transfer to Malonyl-CoA

-CH2CH2CH2C~S-

When C16 stage is reached, instead of transferring to KS,the transfer is to H2O and the fatty acid is released

KS -SH

HSAcetyl-CoA

CoA-SH

-C-CH3

KS S-C-CH3

OKS -SH

CoA-SH

Malonyl-CoA

S -C-CH2-COO-

CO2C=O

CH3-CH -CH2-C-S

OCH3-CH=CH-C-S

OCH3-CH2-CH2-C-S

S-C-CH2-CH2-CH3

NADPH H+

Initiation or priming

Elongation

Fatty Acid SynthaseFatty Acid Synthase

-Ketoacyl-ACP reductase

-Hydroxyacyl-ACP dehydrase

Enoyl-ACP reductase

-Keto-ACP synthase (condensing enzyme)

Malonyl-CoA-ACP transacylase

Acetyl-CoA-ACP transacylase

-Ketoacyl-ACP synthase

Overall ReactionsAcetyl-CoA + 7 malonyl-CoA + 14NADPH + 14H+

Palmitate + 7CO2 + 14NADP+ + 8 HSCoA + 6H2O

7 Acetyl-CoA + 7CO2 + 7ATP 7 malonyl-CoA +7ADP + 7Pi + 7H+

8 Acetyl-CoA + 14NADPH + 7H+ + 7ATP Palmitate + 14NADP+ + 8 HSCoA + 6H2O + 7ADP + 7P

PROBLEM:

Fatty acid biosynthesis takes place in thecytosol. Acetyl-CoA is mainly in the

Mitochondria

How is acetyl-CoA made available to the cytosolicfatty acyl synthase?

SOLUTION:

Acetyl-CoA is delivered to cytosol from the mitochondria as CITRATE

acetyl-CoA

HO-C-COO

Citrate lyase

Acetyl-CoA

COOCH2

HO-C-H

L-malate

C=OCOO

NADPH + H+

L-malate

mitochondria

CytosolPyruvate

Malic enzymeOAA

Acetyl-CoACO2

PyrCO2

Malatedehydrogenase

HS-CoA

Post-Synthesis Modifications

C16 satd fatty acid (Palmitate) is the product Elongation Unsaturation Incorporation into triacylglycerols Incorporation into acylglycerol phosphates

Elongation of Chain (two systems)

HS-CoA

R-CH2CH2CH2C~SCoAO

OOC-CH2C~SCoA

Malonyl-CoA* (cytosol)

R-CH2CH2CH2CCH2C~SCoAO O

O R-CH2CH2CH2CH2CH2C~SCoA

NADPH NADH

- H2O2

NADPH3

Elongation systems arefound in smooth ER andmitochondria

CH3C~SCoA

OAcetyl-CoA(mitochondria)

DesaturationRules:The fatty acid desaturation system is in the smooth membranes of the endoplasmicreticulum

There are 4 fatty acyl desaturase enzymes in mammals designated 9 , 6, 5, and 4 fattyacyl-CoA desaturase

Mammals cannot incorporate a double bondbeyond 9; plants can.

Mammals can synthesize long chain unsaturated fatty acids using desaturation and elongation

Triacylglycerol Synthesis Fatty acyl-CoA DHAP reduction to glycerol-PO4

Glycerol kinase to glycerol-PO4

Two esterifications Diacylglycerol-PO4 intermediate

Triacylglycerol

O-C-RO

R-C-OO

CH2O-C-R

R-C-O-C-HCH2OP

CH2OPC=O

HO-C-HCH2OH

HO-C-HCH2OP

ADP ATP

glycolysis

NADH + NAD+

Glycerol-PO4

glycerol kinase glycerol-PO4

dehydrogenase

Phospholipidbiosynthesis

CH2O-C-R

R-C-O-C-HCH2O-C-R

CH2O-C-R

R-C-O-C-HCH2OH

1,2 Diacylglycerol (DAG)

Triacylglycerol Biosynthesis

Not in adiposetissueR-C~CoA

R-C~CoA

OPhosphatidic acid

Question Can a triacylglycerol (triglyceride) storage

fat be synthesized entirely from glucose, i.e., every carbon in the fat comes from a sugar?

Answer: YES

Metabolism of Phospholipids Phospholipid

phosphorous-containing lipids

fatty acids, a phosphate group, and a simple organic molecule

Glycerolphospholipids (phosphoglycerides) glycerol Sphingolipid

sphingosine

Classification of structural features of glycerolphospholipids

Table 8-2

Phospholipids

hydrophilic head , hydrophobic tail

Membrane

phospholipid bilayer

Glycerolphospholipids

Phosphatidic Acid

Polar componentEster linkage

- or + = choline, serine, ethanolamine, etc

CH2O-C-R

R-C-O-C-HCH2OPO-CH2CH2-N(CH3)3

Phosphatidylcholine or lecithin

PhospholipidBiosynthesis(smooth ER)

- or +O

CH2O-C-R

R-C-O-C-HCH2OP

Glycerophospholipids

Strategy of Glycerophospholipid Biosynthesis Activate diacylglycerol Activate appending moiety (salvage)

RibosePPP-

CH2O-C-R

R-C-O-C-HCH2OP

Eukaryotes

CH2O-C-R

R-C-O-C-HCH2OH

Glycerol-3-PO4Glycerol

Phosphatidic acid

1,2 DAG

choline (CDP-choline)

Glycerol (CDP-diacylglycerol)Serine (phosphatidylethanolamine)

ethanolamine (CDP-ethanolamine)

Inositol (CDP-diacylglycerol)

Cardiolipin (phosphatidylglycerol)

DHAPFA-CoA

1-Acyl-DHAP

1-Acyl-glycerol-3-PO4

CDP-diacylglycerolCTP

CH2CH2(CH3)N3

Cytidine diphosphate (CDP) choline

CH2CH2N3+

Cytidine diphosphate (CDP)

ethanolamine

Regulation of Triacylglecerol Metabolism

Pancreas

primary organ involved in sensing the organism’s dietary and energetic states.

monitoring glucose concentrations in the blood. Low blood glucose stimulates the secretion

of glucagon Elevated blood glucose calls for the

secretion of insulin

Acetaly-CoA carboxylase (ACC) Committed enzyme in fatty acid synthesis

activated by citrate

inhibited by palmitoyl-CoA, long-chain fatty acyl-CoAs

Affected by phosphorylation

glucagon or epinephrine

decreased activity of ACC by phosphorylation

insulin

increases the synthesis of triacylglycerols

Important Derivatives of Unsaturated Fatty Acids- Arachidonic Acid

EICOSANOID FACTS

20-carbon compounds

Include prostaglandins, prostacyclins, thromboxanes, leukotrienes

Physiological effects at very low concentrations

Many of their effects mediated by cyclic AMP or calcium second messengers

Unlike hormones, not transported in the blood

Local mediators that act where synthesized or in adjacent cells

a. the inflammatory response involving primarily the joints (rheumatoid arthritis) and skin (psoriasis);

b. the production of pain and fever;

c. the regulation of blood pressure (vaso-constrictors/dilators) and blood clotting (platelet function);

d. decreased gastric acid secretion (prostacyclins may be an ideal way to control the symptoms of peptic ulcer, but prostanoid synthesis inhibitors, like aspirin, increase acid secretion causing peptic ulcer);

e. the control of several reproductive functions such as the induction of labor and delivery - this has led to the use of PGF2 as a mid-trimester abortifacient drug or as a labor-inducing agent;

f. the regulation of the sleep/wake cycle;

g. hypersensitivity allergic reactions (a primary action of leukotrienes).

The Actions of Prostaglandins and Leukotrienes

esterification

Membrane phospholipids

Dietary linoleic acid

Arachidonic acid

metabolism

Cell Activation Events: mechanical trauma, cytokines growth factors

Anti-inflammatory glucocorticoids

Prostaglandins and thromboxanes(Cyclic/ring product)

Phospholipase A2 (PLA2)

Leukotrienes(Linear product)

Arachidonic acid

Cyclooxygenase(COX)

Lipooxygenase(LOX)

Aspirin, Indomethacin, Ibuprofen NSAIDs

Figure 1. Liberation of arachidonic acid and its metabolism to prostaglandins/ thromboxanes or to leukotrienes

GC induce lipocortin that inhibits PLA2

Aspirin inhibits irreversiblyIndomethacin forms a salt bridge in the binding siteIbuprofen competes for substrate binding

Zyflo competes with AA for binding

LEUKOTRIENE FACTS

leukotriene synthesis inhibited by Zyflo, a lipooxygenase inhibitor

leukotriene action blocked by accolate, a receptor antagonist

peptidoleukotrienes: leukotrienes with short peptides addedcomponents of slow reacting substances of anaphylaxis (SRS-A)anaphylaxis violent (potentially fatal) allergic reaction10,000 times more potent than histamineSRS-A released from lung following immunological stressSRS-A contract smooth muscle causing constriction of bronchiimplicated in hypersensitivity reaction – such as insect sting

Arachidonic Acid (6)derived from membrane phospholipids

central intermediate(Head of pathway)

Prostaglandinendoperoxidesynthase

Xaspirin

indomethacinibuprofen

Cyclooxygenase

Hydroperoxidase

Figure 3. Conversion of arachidonic acid to PGH2

CH2 OH

Figure 4. Structure and mechanism of action of aspirin

CH3CH2 O

AcetylatedCyclooxygenase

(inactive)

Cyclooxygenase(active)

COX-1 VS COX-2 DRUG ACTION

Aspirin:works on both isoformsCOX-1 effect reduces platelet aggregation (TXA2)COX-2 effect reduces inflammationSide effects due to COX-1 inhibition – stomach irritation

Specific COX-2 inhibitorsCelebrex/VioxxTarget inflammatory responseNo COX-1 inhibition to produce aspirin-induced side effects

Aspirin:works on both isoformsCOX-1 effect reduces platelet aggregation (TXA2)COX-2 effect reduces inflammationSide effects due to COX-1 inhibition – stomach irritation

Specific COX-2 inhibitorsCelebrex/VioxxTarget inflammatory responseNo COX-1 inhibition to produce aspirin-induced side effects

Metabolism of Cholesterols

Biosynthesis of Cholesterol Introduction

Functions of cholesterol. Important cell membrane component. Precursor for 3 biologically active compounds.

Bile. Steroid hormones. Vitamin D.

Disease implications. Cardiovascular disease.

Diet control and synthesis manipulation = < heart disorders.

Biosynthesis of Cholesterol Introduction

Disease implications. Gall stones. Steroidogenic enzyme deficiency.

Source of cholesterol. Meat. Eggs. Dairy products. De novo liver synthesis.

Cholesterol Synthesis

Hydroxymethylglutaryl-coenzyme A (HMG-CoA) is the precursor for cholesterol synthesis.

HMG-CoA is also an intermediate on the pathway for synthesis of ketone bodies from acetyl-CoA.

The enzymes for ketone body production are located in the mitochondrial matrix.

HMG-CoA destined for cholesterol synthesis is made by equivalent, but different, enzymes in the cytosol.

CH2 C CH2 C

hydroxymethylglutaryl-CoA

HMG-CoA is formed by condensation of acetyl-CoA & acetoacetyl-CoA, catalyzed by HMG-CoA Synthase.

HMG-CoA Reductase catalyzes production of mevalonate from HMG-CoA.

H3C C CH2 C

CH2 C CH2 C

H2O acetoacetyl-CoA

hydroxymethylglutaryl-CoA

acetyl-CoA HMG-CoA Synthase

The carboxyl of HMG that is in ester linkage to the CoA thiol is reduced to an aldehyde, and then to an alcohol.

NADPH serves as reductant in the 2-step reaction.

Mevaldehyde is thought to be an active site intermediate, following the first reduction and release of CoA.

+ HSCoA

C SCoA

H2C OH

2NADP+

2NADPH

HMG-CoA

mevalonate

HMG-CoAReductase

HMG-CoA Reductase is an integral protein of endoplasmic reticulum membranes.

The catalytic domain of this enzyme remains active following cleavage from the transmembrane portion of the enzyme.

The HMG-CoA Reductase reaction, in which mevalonate is formed from HMG-CoA, is rate-limiting for cholesterol synthesis.

This enzyme is highly regulated and the target of pharmaceutical intervention.

Mevalonate is phosphorylated by 2 sequential Pi transfers from

ATP, yielding the pyrophosphate derivative.

ATP-dependent decarboxylation, with dehydration, yields isopentenyl pyrophosphate.

CH2 OH

CH2 CH2 O P O P O

CH2 O P O P O

ADP + Pi

mevalonate

5-pyrophosphomevalonate

(2 steps)

isopentenyl pyrophosphate

Isopentenyl pyrophosphate is the first of several compounds in the pathway that are referred to as isoprenoids, by reference to the compound isoprene.

isoprene

is o p e n te n y l p y ro p h o s p h a te

H 2 CC

Isopentenyl Pyrophosphate Isomerase inter-converts isopentenyl pyrophosphate & dimethylallyl pyrophosphate.

Mechanism: protonation followed by deprotonation.

CH2 CH2 O P O P O

CH CH2 O P O P O

dimethylallyl pyrophosphate

Prenyl Transferase catalyzes head-to-tail condensations:

Dimethylallyl pyrophosphate & isopentenyl pyrophosphate react to form geranyl pyrophosphate.

Condensation with another isopentenyl pyrophosphate yields farnesyl pyrophosphate.

Each condensation reaction is thought to involve a reactive carbocation formed as PPi is eliminated.

Condensation Reactions

CH2 CH2 O P O P O

CH CH2 O P O P O

CH CH2CH3C

CH CH2 O P O P O

CH2 CH2 O P O P O

CH CH2CH3C

CH CH2CCH2

CH CH2 O P O P O

dimethylallyl pyrophosphate

geranyl pyrophosphate

farnesyl pyrophosphate

Each condensation involves a carbocation formed as PPi is eliminated.

Squalene Synthase: Head-to-head condensation of 2 farnesyl pyrophosphate, with reduction by NADPH, yields squalene.

CH CH2CH3C

CH CH2CCH2

CH CH2 O P O P O

O2 H2O

NADP+ + 2 PP i

2 farnesyl pyrophosphate

squalene 2,3-oxidosqualene lanosterol

Squaline epoxidase catalyzes conversion of squalene to 2,3-oxidosqualene.

This mixed function oxidation requires NADPH as reductant & O2 as oxidant. One O atom is incorporated into substrate (as the epoxide) & the other O is reduced to water.

O2 H2O

H+NADPH

squalene 2,3-oxidosqualene lanosterol

Structural studies of a related bacterial enzyme have confirmed that the substrate binds at the active site in a conformation that permits cyclization with only modest changes in position as the reaction proceeds.

The product is the sterol lanosterol.

2,3-oxidosqualene lanosterol

Squalene Oxidocyclase catalyzes a series of electron shifts, initiated by protonation of the epoxide, resulting in cyclization.

Conversion of lanosterol to cholesterol involves 19 reactions, catalyzed by enzymes in ER membranes.

Additional modifications yield the various steroid hormones or vitamin D.

Many of the reactions involved in converting lanosterol to cholesterol and other steroids are catalyzed by members of the cytochrome P450 enzyme superfamily.

H O H O

lan o ste ro l ch o les te ro l

1 9 s tep s

Regulation of cholesterol synthesis

HMG-CoA Reductase, the rate-limiting step on the pathway for synthesis of cholesterol, is a major control point.

Short-term regulation:

HMG-CoA Reductase is inhibited by phosphorylation, catalyzed by AMP-Dependent Protein Kinase (which also regulates fatty acid synthesis and catabolism).

This kinase is active when cellular AMP is high, corresponding to when ATP is low.

Thus, when cellular ATP is low, energy is not expended in synthesizing cholesterol.

Long-term regulation is by varied formation and degradation of HMG-CoA Reductase and other enzymes of the pathway for synthesis of cholesterol.

Regulated proteolysis of HMG-CoA Reductase:

• Degradation of HMG-CoA Reductase is stimulated by cholesterol, oxidized derivatives of cholesterol, mevalonate, & farnesol (dephosphorylated farnesyl pyrophosphate).

• HMG-CoA Reductase includes a transmembrane sterol-sensing domain that has a role in activating degradation of the enzyme via the proteasome (proteasome to be discussed later).

Long-term regulation is by varied formation and degradation of HMG-CoA Reductase and other enzymes of the pathway for synthesis of cholesterol.

Regulated proteolysis of HMG-CoA Reductase:

• Degradation of HMG-CoA Reductase is stimulated by cholesterol, oxidized derivatives of cholesterol, mevalonate, & farnesol (dephosphorylated farnesyl pyrophosphate).

• HMG-CoA Reductase includes a transmembrane sterol-sensing domain that has a role in activating degradation of the enzyme via the proteasome (proteasome to be discussed later).

Lipid transport

• triacylglycerides, cholesterol, phospholipids

• dietary lipid transport –chylomicron

• endogenous lipid transport (VLDL, IDL, LDL, HDL)

pancreaticlipases

intestinal lumentriacylglycerols FFA + monoacylglycedrols

bile acidscholesterol

micellesmicellesepithelial cellstriacylglycerols

absorbed by intestinal epithelial cells and reconverted to triacylglycerols

•Packaged into chylomicron•Released into lymphatic system and then via capillaries to blood stream

chylomicron

•acted upon by lipases on cell walls of capillaries in tissues

FFA • taken up by tissues

energy production

reconversion to TAGs in adipocytes for storage

hormone sensitive lipasesFFAreleased to circulatory system and combine with albumin fordelivery to tissues

Dietary uptake and distribution of fatty acids

Why do we need lipoproteins? Triacylglycerides (TAGs) + cholesterol (Chol)

are nonpolar molecules → insoluble in H2O

TAG + Chol must be packaged within a polar shell in order to be transported through the blood to the various tissues

This is accomplished by combining nonpolar lipids w/ amphipathic lipids →(a polar water-soluble terminal group attached to an H2O -insoluble hydrocarbon chain)

Lipoproteins & Apolipoproteins

Lipoproteins (LP) function: transport of cholesterol + esterified lipids in

blood structure:

1) polar shell ---single phospholipid (PL) layer: head groups directed outward

-Chol -apolipoproteins2) nonpolar lipid core

-hydrophobic TAG(triacylglycerol)-cholesteryl ester (CE)

apolipoproteins• Provide structural stability to Lp

• Act as cofactors for enzymes involved in plasma lipid and Lp metabolism

• Serve as ligands for interaction w/Lp receptors that help determine disposition of individual particles

There are many types of apolipoproteinsaApoprotein Lipoproteins Function(s)

Apo B-100 VLDL, IDL, LDL 1) Secretion of VLDL from liver 2) Structural protein of VLDL, IDL, and HDL 3) Ligand for LDL receptor (LDLR)

Apo B-48 Chylomicrons, remnants

Secretion of chylomicrons from intestine; lacks LDLR binding domain of Apo B-100

Apo E Chylomicrons, VLDL, IDL, HDL

Ligand for binding of IDL & remnants to LDLR and LRP

Apo A-I HDL, chylomicrons 1) Major structural protein of HDL2) Activator of LCAT

Apo A-II HDL, chylomicrons Unknown

Apo C-I Chylomicrons, VLDL, IDL, HDL

Modulator of hepatic uptake of VLDL and IDL (also involved in activation of LCAT)

Apo C-II Chylomicrons, VLDL, IDL, HDL

Activator of LPL

Apo C-III Chylomicrons, VLDL, IDL, HDL

Inhibitor of LPL activity

Lipoprotein Structure

Lipoproteins• hydrophobic core (TAGS, cholesterol esters)• hydrophilic surface (P-lipids, cholesterol, and

apolipoproteins)

• Functiontransport of lipids in blood

• Types of lipoproteins(classified according to density)

• very low density (VLDL)• intermediate density (IDL)• low density (LDL)• high density (HDL)Protein content increase, lipid decreases as density increases.

% TAGS

% Protein

Chylomicron VLDL IDL LDL HDL85%

Lipoproteins

• Chylomicron:

• 85% TAG, 4% chol., 8% protein•formed in intestinal epithelial cells• deliver exogenous TAGS to tissue• 80 -500nm• ApoCII activates lipases in capillary cell walls releasing FFA to tissue

• chylomicron remnants return to liver where they bind to ApoE receptor and are taken up

• 1/2 life in blood - 4-5 minutes

• VLDL:

• 50% TAGs, 22% choles., 10% protein• 30 -100 nm • formed in liver• deliver endogenous lipids to other tissues(mainly muscle and fat cells)

• ApoCII activates lipases in capillary cell walls releasing FFA to tissue

• converted to IDLs and LDL as lipids are released

• IDL: (31% TAGs, 29% choles., 18% protein) • formed from VLDLs as lipids removed• some IDLs return to liver• rest converted to LDLs by further removal of lipids

Lipoproteins

• LDL: “bad” cholesterol• •10% TAGs, 45% choles., 25% protein• 25 - 30 nm• formed as lipids removed from VLDLs

and IDLs. • all apolipoproteins lost except ApoB100• bind to LDL receptor via ApoB100 and

taken up by endocytosis by hepatic and other tissues (50-75% taken up by liver).

• Primary mode of cholesterol delivery to tissues.• Synthesis of LDL receptor is inhibited by

high levels of intracellular cholesterol and stimulated by low levels of cholesterol.Therefore, cholesterol uptake is closely matched to intracellular cholesterol levels.

• HDL: “good” cholesterol

• 8% TAGs, 30% choles., 33% protein• 7.5 - 10 nm• formed in liver• scavenge cholesterol from cell surfaces

and other lipoproteins and deliver it to liver.• Convert cholesterol to cholesterol ester• bind to “scavenger receptor” on liver cell

surface - cholesterol esters taken up and HDLs released and reenter circulation.

Lipoproteins

Intestine Liver

Dietary lipids

chylomicron

Peripheral tissues

Dietary lipids

chylomicron

TriacylglycerolsFFA

monoacylglycerols

Cholesterol

Cholesterol esters

Triacylglycerolscholesterol

Cholesterol esters

Intestine Liver

Dietary lipids

chylomicron

Peripheral tissues

Dietary lipids

chylomicron

TriacylglycerolsFFA

monoacylglycerols

Cholesterol

Cholesterol esters

Distribution of endogenous lipids The Exogenous Pathway

LPLs activated by ApoCII

Chylomicronremnantsacquire

ApoE, CIIand others

ApoE/LDLRmediated

uptake

Peripheral tissues

TriacylglycerolsFFA

monoacylglycerols

Cholesterol Ester

Cholesterol

Cholesterol esters

Distribution of endogenous lipids The Endogenous Pathway

acquireApoE, CIIand others

LPLs activated by ApoCII

LDLR/ApoE

LDLR/ApoB100

Distribution of endogenous lipids The HDL Pathways

Transport of excess cholesterol from peripheral tissues back to liver for excretion in bile

HDLs act as acceptors for excess chol, Apo, PL derived fromCM, VLDL and LDL

HDLs synthesized by both liver and intestine

Peripheral tissues

TriacylglycerolsFFA

monoacylglycerols

Cholesterol Ester

Cholesterol

Cholesterol esters

IDLsCEs

Distribution of endogenous lipids The HDL Pathways

scavenger receptoruptake of cholesterol

VLDLCholes.

Abnormal Metabolism of Lipoprotein

Hyperlipoproteinemia Genetic diseases

biochem (chapt.5)

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